The Change Of Substance P And It's Receptor And The Effect Of Magnesium On Them In The Midbrain Of Rat Migraine Model

[Objective] With the increasing number of studies on neuropeptide, several kinds of neuropeptide such as substance P (SP), calcitonin-gene-related peptide and neurokinin A, have been proved to correlate with migraine. SP, as a neurotransmitter, plays an important role in sensory information transduction and pain modulation. Currently, SP is considered to elicit diphasic action on sensory information transduction, on one hand, SP is a neurotransmitter of wounding signal introducing into fibers, promoting or facilitating the first synapse of pain sense transmission in cornu dorsale medullae spinalis and spinal nucleus of trigeminal nerve; on the other hand, SP exhibits an analgesic effect in brains, modulates pain sense and reduce susceptibility to pain. Midbrain is of crucial significance in pain modulation, several researches addressing migraine show that numerous SP-positive and SP receptor-positive pericaryons and fibers are observed in periaqueductal gray and rapheal nuclei. However, no quantitative study reports SP mRNA in midbrain. Therefore, the present study adopts real-time quantitative PCR technique, which is considered as the most accurate, most repeatable and generally accepted detection method for nucleic acid molecular from a view of quantitation and qualitation, to conduct a absolutely quantitative study addressing SP mRNA in midbrain of migraine rats. At the same time , Detect the substance P and its receptor protein expression levels in the area with Immunohistochemistry, for discussion Of substance P and its receptor in the pathophysiology of migraine headache in the process of change.Magnesium allows for a better prevention and treatment of migraine by using magnesium in clinical practice. It is assumed that magnesium ion participates in pathophysiological process of migraine through such pathways as promoting cerebral cortex diffusion inhibition, modulating neurotransmitter release, altering cerebral excitability, and influencing platelet function. In the present study, experimental rat models of migraine was established with nitroglycerin by subcutaneous injection, so as to observe behavioral changes and SP and SP receptor expression in midbrain of migraine rats following magnesium intervention.The aim of this study was to investigate:①changes of SP expression in midbrain of migraine rats?②whether behavioral symptom of migraine rats was relieved or exhibited a dose-dependence following magnesium intervention?③whether magnesium inhibiting migraine correlates with SP?[Methods] 1. Grouping, model establishment and interventions : Totally 72 healthy adult Wistar rats were divided into six groups at random, namely control group, migraine group, low- and high-dose of magnesium sulfate treated groups, low- and high-dose of magnesium sulfate control groups, with 12 rats in each group. 2 mL/kg physiological saline was injected into rat cervical and back in the control group, low- and high-dose of magnesium sulfate control groups. Experimental migraine models were established through subcutaneous injection of 10 mg/kg nitroglycerin into rats in the migraine group, low- and high-dose of magnesium sulfate treated groups. The models were considered success upon the appearance of head complaint such as two ears reddish, frequently scratching head with forepaws, increasing number of climbing cage, biting tails, to and fro movement, etc.Five minutes following administration, rats in low dose groups were intraperitoneally injected with 100 mg/kg magnesium sulfate, while those in high dose groups with 300 mg/kg magnesium sulfate. No interventions were given in control group and migraine group.2. Behavioral observation : At 60-90 minutes following nitroglycerin injection, total number of appearing scratching head, climbing cage, biting tails, doing to and fro movement was measured (each symptom accounted for 1 point).3. Detect the substance P and its receptor mRNA expression with real-time PCR:Two hours following nitroglycerin injection, rats were anesthetized with 10% chloral hydrate (0.3 mL/100 g) and then decapitated. The midbrains was isolated and immediately placed in liquid nitrogen, preserved in a refrigerator at -70℃. 50-100 mg of midbrain tissues were added with 1 mL RNAiso Reagent, staying 5 minutes at room temperature, 4℃12 000 g centrifuged for 5 minutes, followed by the supernatant collection. Adding 0.2 mL chloroform, shaking and staying 5 minutes at room temperature, 4℃12 000 g centrifuged for 15 minutes, followed by the supernatant collection. Adding same volume of isopropyl alcohol, staying 10 minutes at room temperature, 4℃12 000 g centrifuged for 10 minutes, followed by the supernatant removal. Adding 1 mL 75% ethanol, 4℃12 000 g centrifuged for 5 minutes, followed by the supernatant removal. The precipitates were dissolved in 100μL RNA-free water. Subsequent to gel electrophoresis, total RNA quality and concentration were determined with ultraviolet spectrophotometer. The ratio of A260/A280 should maintain at 1.9-2.0, RNA concentration was calculated according to the formula: RNA concentration (μg/μL) = A260×attenuation multiple×40/1000. 20μL reaction system comprised RNA 300 ng with Reverse transcriptase. The synthetized cDNA was preserved at -70℃for later use. According to SP,NK1 receptor,NK3 receptor specific primer, the SP,NK1 receptor,NK3 receptor gene was synthetized with PCR apparatus. PCR amplification product was processed into 2% agarose gel electrophoresis, cutting target band, and recovered. The purified target gene was conjugated with pMD-18T carrier and transformed into E.coli DH5αcompetent cell. Subsequent to Ampicillin screening, plasmid extraction commenced. followed by restriction endonuclease HindⅢand BamHⅠdouble digestion, sequencing and identification. The absorbance value of the extracted plasmid at 260 nm was measured, copy number was also calculated. Following sterile water 10-fold serial dilution and subpackage, the standard specimen was preserved at -20℃. Different concentrations of plasmid standard specimens and synthetized cDNA was processed into quantitative PCR. The SP,NK1 receptor,NK3 receptor mRNA content in rat midbrain was calculated based on the melting curve.4. Detect the substance P and its receptor protein expression levels with Immunohistochemistry: Two hours following nitroglycerin injection, rats were anesthetized with 10% chloral hydrate (0.3 mL/100 g) and then Heart perfusion,Slow injection of 10% formaldehyde l00ml fixed,will be a complete ret brain into 4% paraformaldehyde fixed. Conventional dehydration transparent, Low-temperature paraffin-embeded, Sliced. Stained with SP, NK1 receptor, NK3 receptor monoclonal antibody。SP, NK1 receptor, NK3 receptor-Immunoreactive neurons in cell bodies and dendrites were evenly dyed brown。The level of trochlear nucleus in the ventral periaqueductal gray area of Ventral tegmental area and dorsal area in the three horizons, Calculation of the average number of positive cells.5. Statistical analysis: Data were statistically analyzed with SAS 6.12 software (SAS Software Institute, USA). Behavioral scores were expressed as mean±SD, and analysis of variance was applied for comparison among groups. SP mRNA cory number was expressed as mean±SD, and analysis of variance was applied for comparison among groups. The average number of positive cells of the level of trochlear nucleus in the ventral periaqueductal gray area of Ventral tegmental area and dorsal area in the three horizons, were expressed as mean±SD, and analysis of variance was applied for comparison among groups. A level of P < 0.05 was considered statistical significance.[Results] 1. Pain behavioral changes of experimental rats: Statistical results revealed that, nitroglycerin injection yielded to an obviously increment of behavioral scores in rats (P < 0.05). Compared with migraine group, the pain behavior was remarkably decreased in the low- and high-dose of magnesium sulfate treated groups (P < 0.05), especially in the high-dose (P < 0.05).2. The substance P and its receptor mRNA expression levels: SP ,NK1 receptor,NK3 receptor mRNA levels in rat midbrain was calculated according to the standard plot. Statistical analysis results showed that, the migraine group exhibited an obviously lower level of SP mRNA expression than control group (P < 0.05); Compared with migraine group and control group, the SP mRNA expression was enhanced following magnesium sulfate injection (P < 0.05). No significant differences were found among magnesium sulfate administrated groups (P > 0.05). Compared with migraine group and control group, the NK1 receptor mRNA expression was enhanced following high-dose magnesium sulfate administrated groups (P < 0.05). No significant differences were found with NK3 receptor mRNA expression among control group, migraine group, and magnesium sulfate administrated groups (P > 0.05). 3. The substance P and its receptor protein expression levels: Statistical analysis results showed that, Compared with control group, the SP expression was enhanced following high-dose of magnesium sulfate control group (P < 0.05), Compared with migraine group and control group, the SP expression was enhanced following magnesium sulfate injection (P < 0.05). Compared with migraine group and control group, the NK1 receptor expression was enhanced following magnesium sulfate administrated groups (P < 0.05). No significant differences were found with NK3 receptor expression among control group, migraine group, and magnesium sulfate administrated groups (P > 0.05). [Conclusion] 1. In the present study, SYBR GreenⅠreal-time quantitative PCR was successfully established to determine SP,NK1 receptor,NK3 receptor mRNA expression, the standard plot exhibits high linear correlation, sensitivity and repeatability, thus ensuring results more accurate and more reliable.2. Nitroglycerin has been generally acknowledged to establish an experimental animal model of migraine .the successful rat models are similar with the onset manifestations of migraine patients regarding behaviors (ear reddens, scratching head, climbing cage and so on) and pathological biomechanical changes. Model design was in accordance with the principles of standardization, similarity, reproducibility, applicability and economy. The models can be generously replicated in a short term. Therefore it is an optimal candidate model for migraine. However, this model has some limitations of behavioral observation, there is no a quantitative index to evaluate the success of model establishment, or scoring system for behavioral changes, therefore the assessment system on the behaviors should be further improved. 3. SP gene expression was reduced in midbrain when headache occurred.The levels of SP gene expression was decreased in brain of Migraine rat, while the SP was no significant change in protein level expression. we need to be further observed the expression of SP in different periods of migarine, clearly SP in migraine attacks in the process of change.4. varying doses of magnesium sulfate could relieve pain behavior in migraine rats and the therapeutic effect exhibited dose dependence, that is to say, high dose of magnesium sulfate resulted in obvious relieve of pain behaviors. 5. magnesium can cure migraine through the altered expression of SP and NK1 receptor in midbrain.6. magnesium sulfate may prevent migraine through the altered expression of SP and NK1 receptor in midbrain.7. NK3 receptor is not a specificity SP receptor. It is no clear relationship with migraineattacks,as well as the role of prevention and treatment of migraine attacks by magnesium sulfate.